Deledele-tion line 6BS-4 Figure 1 missed a gluten protein band that is present in the other deletion lines of chromosome 6B, even though deletion line 6BS-1 has been reported WGGRC; Figu
Trang 1Open Access
Research article
Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese
Spring deletion lines
Hetty C van den Broeck*†1, Teun WJM van Herpen†1,2,3, Cees Schuit1,
Elma MJ Salentijn1, Liesbeth Dekking4,5, Dirk Bosch1, Rob J Hamer2,
Marinus JM Smulders1,3, Ludovicus JWJ Gilissen1,3 and Ingrid M van der
Meer1,3
Address: 1 Plant Research International, Wageningen UR, PO Box 16, NL-6700 AA Wageningen, The Netherlands, 2 Laboratory of Food Chemistry, Wageningen UR, PO Box 8129, NL-6700 EV Wageningen, The Netherlands, 3 Allergy Consortium Wageningen, PO Box 16, NL-6700 AA
Wageningen, The Netherlands, 4 Leiden University Medical Center, PO Box 9600, NL-2300 RC Leiden, The Netherlands and 5 Dynomics BV,
Erasmus Medical Centre, Department of Immunology, PO Box 82, NL-1400 AB Bussum, The Netherlands
Email: Hetty C van den Broeck* - hetty.busink@wur.nl; Teun WJM van Herpen - teun.vanherpen@wur.nl; Cees Schuit - ceesschuit@bejo.nl;
Elma MJ Salentijn - elma.salentijn@wur.nl; Liesbeth Dekking - e.dekking@erasmusmc.nl; Dirk Bosch - dirk.bosch@wur.nl;
Rob J Hamer - hamer@tifn.nl; Marinus JM Smulders - rene.smulders@wur.nl; Ludovicus JWJ Gilissen - luud.gilissen@wur.nl; Ingrid M van der Meer - ingrid.vandermeer@wur.nl
* Corresponding author †Equal contributors
Abstract
Background: Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals.
In CD patients gluten-derived peptides are presented to the immune system, which leads to a
gluten proteins contain T-cell stimulatory epitopes Gluten proteins are encoded by multigene loci
present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat
(Triticum aestivum) (AABBDD).
Results: The effects of deleting individual gluten loci on both the level of T-cell stimulatory
epitopes in the gluten proteome and the technological properties of the flour were analyzed using
a set of deletion lines of Triticum aestivum cv Chinese Spring The reduction of T-cell stimulatory
epitopes was analyzed using monoclonal antibodies that recognize T-cell epitopes present in gluten
proteins The deletion lines were technologically tested with respect to dough mixing properties
and dough rheology The results show that removing the α-gliadin locus from the short arm of
chromosome 6 of the D-genome (6DS) resulted in a significant decrease in the presence of T-cell
stimulatory epitopes but also in a significant loss of technological properties However, removing
the ω-gliadin, γ-gliadin, and LMW-GS loci from the short arm of chromosome 1 of the D-genome
(1DS) removed T-cell stimulatory epitopes from the proteome while maintaining technological
properties
Conclusion: The consequences of these data are discussed with regard to reducing the load of
T-cell stimulatory epitopes in wheat, and to contributing to the design of CD-safe wheat varieties
Published: 7 April 2009
BMC Plant Biology 2009, 9:41 doi:10.1186/1471-2229-9-41
Received: 5 November 2008 Accepted: 7 April 2009 This article is available from: http://www.biomedcentral.com/1471-2229/9/41
© 2009 Broeck et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2Celiac disease (CD) is a disorder that is characterized by a
permanent intolerance to gluten proteins from wheat, rye,
and barley Over 0.5% of the Western population suffers
from CD, which presents itself by chronic diarrhea,
fatigue, osteoporosis, lymphoma, and several other
clini-cal symptoms after prolonged gluten consumption Until
now, the only treatment is a complete and life long
elim-ination of gluten from the daily diet [1] In the small
intes-tine, several native gluten peptides can bind directly to
specific human leukocyte antigen (HLA)-DQ2 or DQ8
receptors on antigen presenting cells (APCs) However,
after deamidation by tissue transglutaminase (tTG), the
affinity of the peptides for these HLA-receptors is strongly
increased The gluten peptides can be presented by APCs
to gluten-sensitive T-cell lymphocytes leading to the
release of cytokines, which will cause inflammation
reac-tions and result in damaged intestinal villi [2]
Gluten are major storage proteins and have many
interest-ing characteristics for food industrial applications, e.g in
baking bread Gluten proteins can be divided into three
main groups: high molecular weight glutenin subunits
(HMW-GS), low molecular weight glutenin subunits
(LMW-GS), and gliadins The HMW-GS are divided in
x-type and y-x-type subunits [3] The LMW-GS are divided
into B-, C-, and D-type subunits [4] Gliadins are divided
into α/β-, γ-, and ω-gliadins [5] Multiple T-cell activating
gluten peptides were mainly found in α-gliadins, but also
in γ-gliadins and both LMW-GS and HMW-GS [1,2,6,7]
Especially peptides derived from α-gliadins are recognized
by T-cells from most CD patients, while T-cell responses
to γ-gliadins and glutenins are less frequently found
[2,7-10]
Wheat varieties with very low amounts of T-cell
stimula-tory epitopes may be tolerated by many CD-patients
[9,11], while a diet based on wheat varieties reduced in
T-cell stimulatory epitopes may help in the prevention of
CD, as it has been observed that the amount and duration
to gluten exposure is associated with the initiation of CD
[12-14] Breeding for bread wheat (Triticum aestivum) with
less T-cell stimulatory gluten may result, however, in
vari-eties with unwanted loss of technological properties,
because glutenins and gliadins together contribute largely
to dough quality A correct mixture of both glutenins and
gliadins is essential to obtain optimal viscoelastic dough
[15], and the quantity and the size distribution of the
glu-ten proteins are important factors for polymerization
[16,17]
Gluten-encoding genes are located on the three
homoeol-ogous genomes of bread wheat: A, B, and D A few (for
HMW-GS) to a hundred (for α-gliadins) gene copies are
present in hexaploid wheat Sequences of individual gene
copies within the same gluten family, such as the
α-glia-dins, are very similar and may contain multiple and differ-ent T-cell stimulatory epitopes [18] Gluten proteins are encoded by 15 major loci The HMW-GS are encoded by
loci on the long arm of group 1 chromosomes (GluA1, -B1, and -D1) [19] The LMW-GS are mainly encoded by the Glu-3 loci on the short arms of group 1 chromosomes (Glu-A3, -B3, and -D3) [20] and are tightly linked to the loci encoding the γ-gliadins and ω-gliadins (Gli-A1,-B1, and -D1 and Gli-A3, -B3, and -D3) Most α/β-gliadins are
encoded by loci on the short arms of group 6
chromo-somes (Gli-A2, B2, and D2) [21].
In this study, deletion lines of Triticum aestivum cv
Chi-nese Spring (CS) were selected [22-24] These deletion lines are generally lacking one locus containing gluten genes from one of the three homoeologous chromo-somes Here, we explore the feasibility to reduce T-cell stimulatory epitopes in hexaploid bread wheat by screen-ing with epitope-specific monoclonal antibodies [25-27], while maintaining the technological properties
Results
Protein database search
The NCBI protein database search was performed to ana-lyze the number of proteins that contain the different sequences recognized by mAb and T-cells This search pro-vided insight in how many proteins were expected to con-tain the different sequences and which different sequences were present within the proteins The numbers
of protein sequences that contain the various sequences involved in the onset of CD that are recognized by T-cells and mAbs are shown in Table 1 It was observed that the mAb and T-cell minimal sequences were specific for the epitopes in each of the expected protein group, with the exception of the mAb recognizing Glia-α9, whose mini-mally recognized sequence was also present in a number
of γ- and ω-gliadin proteins The sequence recognized by the T-cells was not present within any other protein group except for the α/β-gliadins The minimal sequences recog-nized by mAbs LMW-1 and LMW-2 were more frequently found in the LMW-GS group than the sequence nized by the corresponding T-cells The sequences recog-nized by mAb and T-cells for HMW-glt was present in nearly all HMW-GS protein sequences
SDS-PAGE
To obtain the gluten protein patterns from the CS deletion lines, gluten proteins were extracted and analyzed by SDS-PAGE followed by silver staining Major differences com-pared to CS wild type are indicated by boxes in Figure 1 Differences in gluten protein content compared to CS wild type were mostly observed in the B-, C- type LMW-GS and α/β-, γ-gliadin region Lines with deletions of the short arms of chromosomes 1D were missing several glu-ten protein bands in the ω-gliadin/D-type LMW-GS region The double deletion line, 1BS-19/6DS-4 (Figure
Trang 31), was missing the largest number of gluten protein
bands because of two deletions in gluten encoding
regions Unexpected results were obtained for deletion
line 1BS-18, which is the line with the smallest deletion of
chromosome arm 1BS This line is missing an extra band
compared to the other 1BS deletion lines having larger
deletions This does not fit with reported results on
dele-tion lines [22,23] Deledele-tion line 6BS-4 (Figure 1) missed a
gluten protein band that is present in the other deletion
lines of chromosome 6B, even though deletion line 6BS-1
has been reported (WGGRC; Figure 2C) to contain a larger
deletion than 6BS-4 Deletion line 6BS-4 also contains the
5BS-2 deletion, but, to our knowledge, no gluten protein
locus has ever been identified onto the short arm of
chro-mosome 5B We do not have any explanation for these
discrepancies
Gli-1 deletions
CS deletion lines were analyzed for their contribution to T-cell stimulatory epitopes by using various mAbs recog-nizing different T-cell epitopes In Figure 2A, immunoblot results are presented using mAbs Glia-α9 and Glia-α20 for
deletion lines of the short arm of chromosomes 1 (Gli-1) and 6 (Gli-2) Major differences, compared to CS wild
type, are indicated with arrowheads Deletion lines 1AS-3 and 1AS-1 were missing one gluten protein band by using mAb Glia-α9 and no gluten protein bands by using mAb Glia-α20 (Figures 2A and 2B) This suggests that this miss-ing gluten protein only contains the epitope sequence rec-ognized by mAb Glia-α9 and the loci encoding these gluten protein map to bin 1AS3-0.86–1.00 (the terminal 14% of chromosome arm 1AS) (Figure 2C) All five dele-tion lines of the short arm of chromosome 1B (Figure 2A) lacked one gluten protein band by using mAb Glia-α9 and
no gluten protein band by using mAb Glia-α20 The dou-ble deletion line 1BS-19/6DS-4 (Figure 2A) was missing two extra bands using mAb Glia-α9 and four by using mAb Glia-α20, which is caused by the 6DS-4 deletion Two gluten protein bands were recognized by both mAbs Glia-α9 and Glia-α20 All 1BS deletion lines (Figure 2A) lacked the same gluten protein band recognized by mAb Glia-α9 and because of that the loci encoding correspond-ing gluten protein map to bin 1BSsat18-0.50–1.00 (Figure 2C) All three deletion lines of the short arm of chromo-some 1D (Figure 2A) lacked four gluten protein bands by using mAb Glia-α9 and two gluten protein bands by using mAb Glia-α20 These missing protein bands correspond
to the boxed (missing) proteins in Figure 1 One gluten protein band did not completely disappear by using mAb Glia-α9 This is probably because of the presence of gluten
Table 1: Results of database search for sequences recognized by mAbs and T-cells.
Protein groups
The number in each cell represents the presence of the recognized sequence by mAb or T-cell within a protein group '-' in a cell means that the sequence was not present.
SDS-PAGE analysis of prolamin extracts from Chinese Spring
deletion lines
Figure 1
SDS-PAGE analysis of prolamin extracts from
Chi-nese Spring deletion lines CS: ChiChi-nese Spring wild type
Boxes indicate differences in protein bands
ω-gliadins D-type LMW-GS
B-, C-LMW-GS α/β-, γ-gliadins
HMW-GS
kDa
200.0
116.3
97.4
66.2
45.0
31.0
CS 1
CS 6
Trang 4proteins from different loci but having the same
molecu-lar weights, therefore becoming visible only as one gluten
protein band The loci encoding the recognized gluten
proteins map to bin 1DS5-0.7–1.0 (the terminal 30% of
1DS) (Figure 2C) The two gluten protein bands
recog-nized by mAb Glia-α20 were the same as recogrecog-nized by
mAb Glia-α9
Gli-2 deletions
When analyzing CS deletion lines that are lacking parts of
the short arm of chromosome 6, deletion line 6AS-1
(Fig-ure 2A) lacked one gluten protein band in
immunoblot-ting using mAb Glia-α9 and two bands by using mAb
Glia-α20 Deletion line 6BS-4 (Figure 2A) lacked one glu-ten protein band by using mAb Glia-α9, but this was not the case for the other two 6BS deletion lines, 6BS-1 and 6BS-5 (Figure 2A), which is not consistent with the reported sizes of the deletions In the 6BS deletion lines,
no changes were observed in gluten protein bands com-pared with CS wild type by using mAb Glia-α20 (Figure 2B) These results suggest that the short arm of chromo-some 6B encodes no gluten proteins containing T-cell stimulatory epitopes recognized by both mAbs Glia-α9 and Glia-α20, at least not mapping to bin 6BS-0.25–1.00 (terminal 75% of 6BS) (Figure 2C) Deletion line 6DS-2, the line with the largest deletion (Figures 2A and 2B)
Immunoblot analysis of Chinese Spring deletion lines of the short arm of chromosome 1 and 6
Figure 2
Immunoblot analysis of Chinese Spring deletion lines of the short arm of chromosome 1 and 6 (A) Using mAb
Glia-α9 (B) Using mAb Glia-α20 CS: Chinese Spring wild type Arrowheads indicate absent protein bands (C) Physical maps
of the short (S) arms of wheat chromosomes 1A, 1B, 1D, 6A, 6B, and 6D from centromer to telomeric ends (Wheat Genetic and Genomic Resources Centre, Kansas State University, USA) Arrows on the right of each chromosome indicate the dele-tion lines with their breakpoint (indicated as fracdele-tion length from the centromer) The banding patterns within the chromo-somes are according to Gill et al [24]
CS
CS
60 50
40
30
A
B
kDa 60 50
40
30
C
satellite
satellite
Trang 5lacked two gluten protein bands recognized by mAb
Glia-α9 and four bands by mAb Glia-α20 One gluten protein
band has not completely disappeared probably because of
the presence of different gluten proteins having the same
molecular weight within one gluten protein band The
same gluten protein bands are also absent in the double
deletion line 1BS-19/6DS-4 (Figure 2A) These missing
protein bands correspond to the boxed (missing) proteins
in Figure 1 Hence, the loci encoding these gluten proteins
map to bin 6DS4-0.79–0.99 (Figure 2C)
Glu-3 deletions
The immunoblot results using mAb LMW-2 for the
dele-tion lines of the short arm of chromosome 1 are shown in
Figure 3 One band was observed in all the deletion lines
and in CS wild type without significant differences
Immunoblot results using mAb LMW-1 showed similar
patterns (results not shown)
Glu-1 deletions
Within the protein database, nearly all HMW-GS had
epitope sequences recognized by mAb HMW-glt The
immunoblot results for the deletion lines of the long arm
of chromosome 1 using the mAb recognizing HMW-glt
are shown in Figure 4A In CS wild type, all four HMW
glutenin subunits were detected No contribution to
HMW-GS was observed for the long arm of chromosome
1A, as expected for a transcriptional silent locus Two
HMW-GS, 1Bx7 and 1By8, were absent in deletion lines
1BL-1 and 1BL-6 This suggests that the locus encoding
HMW-GS 1Bx7 and 1By8 map to bin 1BL1-0.47–0.69
(Figure 4B) The two HMW-GS, 1Dx2 and 1Dy12, were
absent in deletion line 1DL-4 This suggests that the loci
encoding HMW-GS 1Dx2 and 1Dy12 map to bin
1DL4-0.18–0.41 (Figure 4B)
Rheological parameters of Chinese Spring deletion lines
The lines with the largest deletions from chromosomes 1
and 6, according to our results, were used for
technologi-cal testing Parameters among flours of different deletion
lines are presented in Figure 5 and in the Additional file 1: Rheological parameters
Total protein content in flour (% w/w) of all deletion lines was higher compared to CS wild type flour Especially pro-tein content in flour of line 6AS-1 was high (20.5%), fol-lowed by protein content in flour of deletion line 1BS-19/ 6DS-4 (18.6%)
The glutenin macro polymer (GMP) content expressed as volume per mg protein was decreased in deletion line 1BL-1 and was nil in deletion line 1DL-4 (Figure 5 and Additional file 1: Rheological parameters) GMP repre-sents the highly aggregated glutenin protein network that
is the prime determinant of dough elastic properties A decrease in GMP is therefore expected to lead to a decrease
in dough strength [28-30] Because of the low amount of GMP present in flour of the deletion lines 1BL-1 and
1DL-4, it was impossible to estimate glutenin particle sizes for these lines Flours of the two deletion lines, 1BS-10 and 6DS-2, showed a small decrease in GMP volume For all other deletion lines, the GMP volume was increased Glutenin particle size is a predictor of dough mixing prop-erties [31] Average glutenin particle size was increased in flours of deletion line 1AL-1 and 6AS-1 In deletion lines
Immunoblot analysis of Chinese Spring deletion lines of the
short arm of chromosome 1, using mAb LMW-2
Figure 3
Immunoblot analysis of Chinese Spring deletion lines
of the short arm of chromosome 1, using mAb
LMW-2 CS: Chinese Spring wild type.
kDa
60
50
40
30
CS
Immunoblot analysis of Chinese Spring deletion lines of the long arm of chromosome 1
Figure 4 Immunoblot analysis of Chinese Spring deletion lines
of the long arm of chromosome 1 (A) Using mAb
HMW-glt CS: Chinese Spring wild type (B) Physical maps of the long (L) arms of wheat chromosomes 1A, 1B, and 1D from centromer to telomeric ends (Wheat Genetic and Genomic Resources Centre, Kansas State University, US) Arrows on the right of each chromosome indicate the dele-tion lines with their breakpoint (indicated as fracdele-tion length from the centromer) The banding patterns within the chro-mosomes are according to Gill et al [24]
B
centromer centromer centromer
A
kDa
120 84
CS
Trang 66DS-2, 6BS-1, 1BS-10, 1BS-19/6DS-4 and 1AS-1 the
aver-age particle size was decreased compared to CS wild type
Dough made from flours of the two deletion lines 1BL-1
and 1DL-4, lacking HMW-GS, showed a significant
decrease in dough development time (DDT) (Figure 5 and
Additional file 1: Rheological parameters) Dough made from all other deletion lines showed increase in DDT, especially the lines with deletions of the short arm of chromosome 6 (6AS-1, 1BS-19/6DS-4, 6DS-2, and 6BS-1)
and 1AS-1 Deletions of the Gli-2 loci seem to have a
sub-stantial effect on increasing DDT
Bandwidth at peak resistance (BWPR) is a measure of dough stability The BWPR was slightly decreased for dele-tion line 1DL-4 and was increased for all other deledele-tion lines compared to CS wild type dough (Figure 5 and Addi-tional file 1: Rheological parameters) The BWPR was especially high for deletion lines 6AS-1 and
1BS-19/6DS-4 It is relevant to note that these are the same deletion lines having the highest protein content in flour
Dough elasticity, indicated by relaxation half time (T1/2), was decreased in flours of deletion lines 1BL-1 and
1DL-4, which lack HMW-GS, and in deletion lines 6BS-1 and 6DS-2 (Figure 5 and Additional file 1: Rheological param-eters) In contrast, deletion lines 1BS-19/6DS-4 and
dough [32,33]
To summarize, in Figure 6 immunoblots are shown for Chinese Spring wild type and the gliadin proteins reacting with mAbs Glia-α9 and Glia-α20 are numbered In Table
2 the relation is shown of these proteins together with their bin-location on the chromosomes and the rheologi-cal parameters if these proteins are missing in the deletion lines Deletions of the long arms of chromosome 1A, 1B, and 1D are not included because the HMW-GS encoded
by the loci on these arms (1BL and 1DL) seem to be required for good technological properties
Discussion
In this study, we examined the possibilities to develop a bread wheat variety with both reduced levels of T-cell stimulatory epitopes and good technological properties
We used a set of Chinese Spring deletion lines that lack different gluten protein-encoding loci from the group 1 and 6 chromosomes to determine whether reduction in T-cell stimulatory epitopes can be achieved by removal of certain gluten protein encoding genes with minimal effect
on the technological properties of bread wheat Many
cytogenetic resources have been developed in T aestivum
cv Chinese Spring, which is considered as a model variety for hexaploid wheat However, differences among varie-ties may exist
CD immunogenic epitopes
On the short arm of the group 6 chromosomes, the gluten loci that encode α-gliadins are located The α-gliadins are considered the most immunogenic concerning both the adaptive immune response and the innate immune
Rheological parameters tested for Chinese Spring deletion
lines
Figure 5
Rheological parameters tested for Chinese Spring
deletion lines All technological measurements were
per-formed in duplicate, except the relaxation test (T1/2) for
dele-tion lines 1DL-4, 6AS-1, 6DS-2, and 6DS-4/1BS-19 Error
bars represent the standard error 'NA' means not analyzed
for particle surface area (D3,2) because the amount of GMP
was too low
0
1
2
3
4
5
0
5
10
15
20
25
30
35
40
45
T ½
0
10
20
30
40
50
60
70
0
4
8
12
16
20
24
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
16
18
20
WT 1AL-1 1BL-1 1DL-4 1AS-1 1BS-10 1DS-1 6AS-1 6BS-1 6DS-2 1BS-19/
6DS-4
Trang 7response [2,8,10,11] We observed that the locus on the
short arm of chromosome 6D, mapped to bin 6DS-0.45–
0.99, is responsible for most of the T-cell stimulatory
α-gliadin proteins These results are in agreement with the
results obtained by Molberg et al [34] who showed no
decrease in response of DQ2-α-II T-cells for deletion line
6DS-6 and a significant decrease in T-cell response for
deletion lines 6DS-4 and 6DS-2 In addition, results are in
agreement with results of Van Herpen et al [18], based on
relative presence of CD-epitopes in α-gliadin ESTs from
the three homoeologous loci, and with results of Salentijn
et al [35] on the presence in cDNAs from two hexaploid
and two tetraploid cultivars When using mAb Glia-α20 in
immunoblotting also two gluten protein bands were
stained that were encoded by the short arm of
chromo-some 1D We tentatively assign these as ω-gliadins/D-type
LMW-GS containing the mAb Glia-α20 sequence Only a
few ω-gliadin proteins have been sequenced so far
because they are difficult to clone due to the presence of
large repetitive domains [36] It has been shown that
ω-gliadins may have epitopes that are involved in
gluten-sensitive response of CD patients [37,38] The α-gliadins
encoded by chromosome 6 seem to be related to gliadins
encoded by chromosome 1 from which they might have
originated through gene duplication and/or translocation
[39,40] Analysis of the minimal sequence recognized by
mAb Glia-α9 indicated that this sequence also occurs in
some γ- and ω-gliadins Indeed, mAb Glia-α9 recognized gluten protein bands that disappeared in deletion lines of the short arm of chromosome 1A, 1B, and 1D (where γ-and ω-gliadin encoding genes are located) We observed that genes mapped to bin 1DS-0.48–1.00 had the highest contribution to the number of T-cell stimulatory epitopes
Technological properties
Studies have shown that the technological parameters of wheat flours are influenced by alleles encoding different HMW-GS [41-44], LMW-GS [45,46], and gliadins [47] Deleting parts of the short arm of chromosome 1A resulted in an increased dough development time (DDT) and volume of glutenin macro polymer (GMP) A decrease in LMW-GS or gliadins results in a relative increase of ratios for HMW-GS/LMW-GS or glutenins/ gliadins Such a change was suggested to increase dough strength [15,16] Indeed, we found that removal of the locus from the short arm of chromosome 1A resulted in increased dough elasticity In the deletion lines 1AS-1 and 1DS-1, higher GMP volumes were observed, while in dele-tion line 1BS-10 a decreased GMP volume was found together with decreased DDT On chromosome 1B, also a
Glu-B2 locus is located encoding a B-type LMW-GS [48,49] and a Glu-B3 locus is located encoding two tightly
linked genes for an ω-gliadin and a B-type LMW-GS [50] This suggests that LMW-GS encoded by these loci are important for the formation of the GMP [51,52] Removal
of the loci could affect the ratios for HMW-GS/LMW-GS or glutenins/gliadins Chromosome 1D encodes a D-type LMW-GS containing a single cysteine residue and there-fore may act as a chain terminator [53,54] The absence of the protein could increase the GMP volume in deletion line 1DS-1 It would be expected that the GMP volume would decrease in deletion line 1AS-1 because of removal
of the locus encoding major LMW-GS We observed, how-ever, that no T-cell stimulatory epitopes present in
GS disappeared from the immunoblot using mAbs
LMW-1 and LMW-2, which is possible if expression from the deleted locus is compensated for by the other two loci present on the homoeologous chromosomes, for example
by a higher expression of Glu-B3 Compensation behavior
of storage protein synthesis in wheat was observed by Wieser et al [55] after inhibition of the expression of α-gliadins by RNA interference (RNAi) Also Gil-Humanes
et al [56] recently observed while RNAi reduced the pro-portion of γ-gliadins by 55–80% and α-gliadins by 63%, this did not lead to similar reduction in proteins detected
by the sandwich ELISA using the R5 monoclonal anti-body The R5 assay was, however, developed for the detec-tion of gluten proteins from different sources and not optimized to detect T-cell stimulatory gluten proteins [57] Hence, although the R5 assay is currently considered the standard test for identification of gluten
contami-Numbering of protein bands reacting with mAbs Glia-α9 and
Glia-α20 in Chinese Spring wild type
Figure 6
Numbering of protein bands reacting with mAbs
Glia-α9 and Glia-α20 in Chinese Spring wild type
Immunoblots of Chinese Spring wild type using mAbs Glia-α9
(left) and Glia-α20 (right)
Glia- α9 Glia-α20
Chinese Spring
1 2 3 4 5 6 7 9 8 10 11 12
Trang 8nants, we regarded this test unsuitable in the context of
this study
With respect to technological properties, deletion line
6AS-1 showed an increase in GMP volume and a strong
increase in glutenin particle size In contrast, deletion
lines 6BS-1 and 6DS-2 showed a decrease in glutenin
par-ticle size and a decrease in GMP volume for deletion line
6DS-2 Gliadins of the α- and γ-type have been identified
to contain an extra cysteine residue that makes them act as
chain terminators We suggest that the short arm of
chro-mosome 6A in CS is encoding a chain terminating
α-glia-din The lower content of chain terminators could account
for a larger size of glutenin particles as observed in
dele-tion line 6AS-1 Because of compensadele-tion, deledele-tions of the
short arm of chromosome 6B and 6D could lead to an
increased expression of chain terminating α-gliadins
encoded by the short arm of chromosome 6A and result
in observed smaller glutenin particle sizes The deletions
of the short arm of chromosome 6B and 6D resulted in stronger dough as shown by increased DDT This effect on dough strength is expected because a decrease in α-glia-dins results in a relative increase of the glutenin/gliadin ratio The GMP volume of flour from deletion line 6DS-2 was decreased, which indicates weaker dough, whereas the DDT was increased, which indicates stronger dough Because of this effect, the decrease in GMP volume in dele-tion line 6DS-2 resulted in decreased elasticity rather than decreased dough strength
We observed that technological properties of flour from deletion lines were strongly affected by the removal of the different HMW-GS with the strongest effect in deletion line 1DL-4 Dough strength (as expressed as DDT and
strongly decreased, which is in agreement with published results [15,58,59] Deletion of the locus on the long arm
of chromosome 1A resulted in some increase in dough
Table 2: Bin location of gliadin proteins and effect on rheological parameters if absent in deletion lines.
Gliadin protein
bands in CS
in flour (%)
(μl/mg)
Particle surface
1.00
1.00
1.00
1.00
1.00
1.00
0.99
0.99
0.99
0.99
1.00
1.00
Protein bands are numbered as shown in Figure 6 and their bin location is determined Bin location is linked to rheological parameters Results are compared to Chinese Spring, which is set at "0" "+" in cell means value is up to 50% higher "++" in cell means value is between 50 to100% higher
"-" in cell means values is up to 50% lower " " in cell means value is between 50 to100% lower "Yes" or "no" in cell means the reaction with the mAb.
Trang 9strength (DDT and GMP volume) and elasticity (T1/2) In
addition, glutenin particle sizes were significantly
increased Both the x-type and y-type encoding genes of
CS at Glu-A1 are silent [19] In most studies, the silent
locus at Glu-A1 was not found to be important to
deter-mine dough strength compared to non-silent loci [45,46],
so the effect of deletion of the long arm of chromosome
1AL might be because of the absence of other gene
prod-ucts Based on these results, the Glu-1 loci of CS are
con-sidered inappropriate as a focus to breed for wheat with
less T-cell stimulatory epitopes if technological properties
are to be preserved
Conclusion
A strategy to breed for bread wheat with less T-cell
stimu-latory gluten epitopes while retaining technological
prop-erties is feasible by focusing on eliminating genes present
on the short arms of chromosome 1D and 6D This will
result in a wheat variety with highly decreased T-cell
stim-ulatory epitopes However, eliminating genes might
decrease dough elasticity because of a changed ratio in
glutenin and gliadin proteins This ratio could be
com-pensated for by the addition of monomeric proteins with
no T-cell stimulatory to the flour, for example from safe
sources like oats, or by the introduction through breeding
or genetic modification of CD-safe gliadin genes In
addi-tion, wheat varieties with limited but not complete
reduced levels of T-cell stimulatory epitopes may still
con-tribute to lower the gluten load for the entire population
and it may reduce the development of CD in a number of
potential patients
Methods
Wheat materials
From the Wheat Genetic & Genomic Resources Center
(WGGRC) Kansas State University, USA
http://www.k-state.edu/wgrc/Germplasm/Deletions/del_index.html,
twenty-six T aestivum Chinese Spring deletion lines were
selected as described [22-24] The deletion lines had
par-tial deletions of the long and short arms of chromosomes
1 and 6, which was characterized by cytogenetics (Figures
2C and 4B) One line contained deletions of both the
short arm of chromosome 1 and chromosome 6 (1BS-19/
6DS-4, Figure 2C) All deletion lines were grown in
con-tainment glasshouses No morphological differences were
observed Seeds were harvested from mature wheat plants
Database search for the specificity of the sequences
recognized by mAbs compared to T-cell epitopes
The frequency of occurrence of known T-cell epitopes
involved in the onset of CD was analyzed by searching
within the National Center for Biotechnology
Informa-tion (NCBI) database From the NCBI protein database
http://www.ncbi.nlm.nih.gov/ five different groups of
gluten protein sequences were extracted and subsequently
converted into FASTA formats, using the following search
queries: 'alpha gliadin', 'gamma gliadin', 'omega gliadin' 'D-type LMW-GS', 'LMW glutenin', and 'HMW glutenin'
All non-Triticum, non-Aegilops entries, and sequences
con-taining less than 100 amino acids were removed For the 'HMW glutenin' group only full size sequences were ana-lyzed The obtained protein sequences were aligned using ClustalW to validate if the correct groups were assigned to the sequences Within the 'gamma gliadin' group, four sequences (AAA34286, P04729, P04730, and AAA34285) were more similar to LMW glutenins and were transferred
to the 'LMW glutenin' group In the 'omega gliadin/D-type LMW-GS' group, one sequence (ABI20696) was specific for the 'alpha gliadin' group and was transferred to the 'alpha gliadin' group The sequences in the five estab-lished groups were analyzed for the different minimal rec-ognition sequences of mAbs and T-cells [25] No mismatches were allowed Scores were expressed as the number of sequences and as the percentage of the sequences in the established group that contained one or more recognition sequences The T-cell minimal recogni-tion sequences used in the analyses were: Glia-α9 (PFPQPQLPY), Glia-α20 (FRPQQPYPQ), LMW-glt (PFSQQQQSPF), HMW-glt (QGYYPTSPQ) and mAb minimal recognition sequences used were: Glia-α9 (QPF-PQPQ), Glia-α20 (RPQQPYP), 1 (PPFSQQ),
LMW-2 (QSPF), HMW-glt (QGQQGYYP) [LMW-25-LMW-27,60]
Extraction of gluten proteins
Gluten proteins were extracted from wheat grains accord-ing to Van den Broeck et al [61] Grains were ground in
an analytical mill (A 11 Basic, IKA-Werke) and sieved through mesh (0.5 mm) Gluten proteins were extracted from 50 mg wheat flour by addition of 0.5 ml of 50% (v/ v) aqueous iso-propanol with continuous mixing (MS1 Minishaker, IKA Works, Inc.) at 1000 rpm for 30 min at room temperature, followed by centrifugation at 10,000 rpm for 10 min at room temperature The residue was re-extracted twice with 50% (v/v) aqueous iso-propanol, 50mM Tris-HCl, pH 7.5 containing 1% (w/v) DTT, for 30 min at 60°C with mixing every 5 to 10 min followed by centrifugation at 10,000 rpm for 10 min at room temper-ature After addition of each next extraction solution, the
FP220A Instrument for 10 sec at 6.5 m/sec followed by sonication for 10 min in an ultrasonic bath (Branson
3510, Branson Ultrasonics Corporation) The three obtained supernatants were combined and considered the gluten protein extract The protein content was quantified using the Biorad Protein Assay (Bio-Rad Laboratories), based on the Bradford dye-binding procedure, according
to manufacturer's instruction with BSA as a standard
SDS-PAGE
Gluten proteins were separated on SDS-PAGE gels (10%) using a SE260 mighty small II system (GE Healthcare, UK) SDS-PAGE was followed by immunoblotting or by
Trang 10silver staining [62] with some modifications Gels were
fixed in 50% (v/v) ethanol/10% (v/v) acetic acid in water
for 30 min Then, gels were washed in 5% (v/v) ethanol/
1% (v/v) acetic acid in water for 10 min, followed by three
times washing for 5 min in MilliQ water Gels were
sensi-tized in 0.02% (w/v) sodium thiosulfate for 1 min and
again washed three times for 30 sec in MilliQ water Gels
were incubated in 0.1% (w/v) silver nitrate for at least 20
min After this incubation, gels were rinsed 2 times for 5
sec in MilliQ water and developed in 6% (w/v) sodium
carbonate containing 0.05% (v/v) formaldehyde (37%)/
0.4‰ (w/v) sodium thiosulfate Development of staining
was stopped by addition of 5% HAc/water
Immunoblotting
Proteins were blotted onto nitrocellulose (0.2 μm,
Bio-Rad Laboratories), in buffer omitting methanol, using a
Mini Trans-Blot Cell (Bio-Rad Laboratories) at 100 V for 1
hour Blots were incubated and visualized as described
[63] using mAbs specific for T-cell stimulatory epitopes
against Glia-α9 [26,60], Glia-α20 [25,60], GLT-156
(LMW-1 and LMW-2) [27,60], HMW-glt [26,60]
Mono-clonal Ab binding was visualized by staining for alkaline
phosphatase, using Nitro Blue tetrazolium (NBT) and
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma)
Quadrumat milling
To obtain white wheat flour, wheat kernels (total weight
ranging 7.6–36 g) were milled using a Quadrumat JR
(Brabender, Germany) Kernel moisture was adjusted to
16.5% Bran was separated from endosperm flour by
siev-ing through mesh (150 μm) After sievsiev-ing the average
yield was 50% (w/w), noting that samples 6AS-1 and
6DS-2 had a typically higher flour yield of 64% and 60%,
the other samples ranged from 43% to 51%
Total protein content in flour
Flour protein content was estimated by the Dumas method
[64] using an NA2100 Nitrogen and Protein Analyzer
(Ther-moQuest-CE Instruments, Rodeno, Italy) The Dumas
method is based on the measurement of total nitrogen in the
sample (N × 5.7) Methionine was used as a standard
Isolation of glutenin macro polymer from flour and
glutenin particle size analysis
Dough strength is correlated to the amount of the
glute-nin macro polymer (GMP) and to the size of gluteglute-nin
par-ticles Glutenin macro polymer was isolated by dispersing
wheat flour in 1.5% (w/v) SDS followed by
ultracentrifu-gation as described [29] Fresh GMP from flour was
dis-persed in 1.5% (w/v) SDS (10 ml) by rotating overnight at
room temperature Particle size distributions were
meas-ured using a Mastersizer 2000 (Malvern Instruments, UK)
The laser diffraction pattern obtained with the instrument
was correlated to the particle size distribution based on Fraunhofer theory, assuming a spherical particle shape The range of the instrument was 0.02–2000 μm Disper-sions of GMP were transferred to the water filled sample vessel at an obscuration of approximately 8% The surface
distribu-tion data for comparisons Further details of this method are described by Don et al and Wang et al [31,65]
Mixing experiments
Dough strength was determined using a micro-Mix-ograph A 2 g Mixograph (National Manufacturing Co., USA) pin-mixer was used to analyze the mixing properties
of the different flour samples Mixing was performed at 20°C Water was added according to the Plastograph method (ICC 115/1 (ICC, 1992) [66] Dough contained 2% (w/w) sodium chloride (Merck, Germany) Band-width at peak resistance (BWPR) in percentages and dough development time (DDT) in minutes were used from the midline analysis for comparison
Flow-relaxation measurements
Relaxation tests were performed to study dough elasticity Longer relaxation half times indicate more elastic dough behavior [32,33] Dough was mixed to peak in the 2 g Mixograph pin-mixer, carefully removed from the mixer and transferred to the Bohlin VOR rheometer (Bohlin Instruments, Sweden) Flow-relaxation measurements were performed using an aluminum grooved plate geom-etry with a cross-section of 30 mm and a gap of 1 mm [33] Moisture loss from the dough piece was prevented using paraffin oil The actual measurement was performed after an equilibration time of 30 min to allow appropriate release of dough stress The measuring temperature was 20°C During measurement, the sample was deformed to
was kept constant and the subsequent decrease of stress of the dough was recorded as a function of time The time necessary for the dough to relax to a stress of 50% of the initial stress, recorded directly after stopping deformation, was used as the flow-relaxation half time (T1/2)
Authors' contributions
IMM and MJMS initiated this study MJMS and CS selected Chinese Spring deletion lines CS and HCB extracted glu-ten proteins from deletion lines EMJS and TWJMH per-formed data base search LD raised monoclonal antibodies HCB performed SDS-PAGE and immunoblot-ting TWJMH performed technological tests IMM super-vised HCB and CS RJH, DB, MJMS, and LJWJG supersuper-vised TWJMH HCB, TJWMH, RJH, MJMS, LJWJG, and IMM contributed to writing the manuscript EMJS, LD, and DB gave editorial comments All authors read and approved the final manuscript